Most diagnostic tests for periodontal pathogens have focused on enumeration of the major gram-negative species associated with diseased sites, although both gram-negative and gram-positive bacteria have been implicated as etiological agents of periodontal disease. The long-term objective of this project is to develop a DNA probe-based assay for detection and quantitation of two gram-positive bacteria which have been associated with sites of periodontal destruction, Peptostreptococcus micros and Streptococcus intermedius. The specific aims of Phase I of the project are (1) to prepare genomic DNA from P. micros and S. intermedius, (2) to develop a sample preparation protocol which assures rapid and complete lysis of gram-positive bacteria before immobilization of DNA on nitrocellulose, (3) to determine the sensitivity and specificity of whole genomic probes for P. micros and S. intermedius, (4) to construct genomic libraries for P. micros and S. intermedius, and (5) to identify clones from the genomic libraries which can be used as highly sensitive and species-specific DNA probes. Many genomic DNA fragments will be screened by Southern blot analysis to identify those which do not cross-react with genomic DNA probes from closely related species. Achievement of these Phase I goals will allow the validation (in Phase II) of DNA probe based assays for the detection of P. micros and S. intermedius in subgingival plaque samples. Inclusion of these assays in a diagnostic test panel for periodontal pathogens will provide clinicians with a more comprehensive assessment of the microbial status of diseased sites. PROPOSED COMMERCIAL APPLICATION: Upon completion of this project, highly sensitive and specific assays for P. micros and S. intermedius will be added to the diagnostic test panel for periodontal pathogens currently marketed by OmniGene's clinical reference laboratory. With a more comprehensive assessment of the microbial status of diseased sites, clinicians will be better prepared to design and monitor the effectiveness of antimicrobial therapies. The expanded test will also be useful to clinical researchers evaluating the efficacy of new therapies for periodontal disease.